Lactoyl leucine and isoleucine are bioavailable alternatives for canonical amino acids in cell culture media.

Increasing demands for protein-based therapeutics such as monoclonal antibodies, fusion proteins, bispecific molecules, and antibody fragments require researchers to constantly find innovative solutions. To increase yields and decrease costs of next generation bioprocesses, highly concentrated cell culture media formulations are developed but often limited by the low solubility of amino acids such as tyrosine, cystine, leucine, and isoleucine, in particular at physiological pH. This study sought to investigate highly soluble and bioavailable derivatives of leucine and isoleucine that are applicable for fed-batch processes. N-lactoyl-leucine and N-lactoyl-isoleucine sodium salts were tested in cell culture media and proved to be beneficial to increase the overall solubility of cell culture media formulations. These modified amino acids proved to be bioavailable for various Chinese hamster ovary (CHO) cells and were suitable for replacement of canonical amino acids in cell culture feeds. The quality of the final recombinant protein was studied in bioprocesses using the derivatives, and the mechanism of cleavage was investigated in CHO cells. Altogether, both N-lactoyl amino acids represent an advantageous alternative to canonical amino acids to develop highly concentrated cell culture media formulations to support next generation bioprocesses.

Keto leucine and keto isoleucine are bioavailable precursors of their respective amino acids in cell culture media.

Highly concentrated cell culture media formulations are essential to develop next generation bioprocesses used to produce therapeutic monoclonal antibodies, fusion proteins, bispecific molecules and mAb fragments. Although cysteine/cystine and tyrosine are the first components preventing the development of highly concentrated complex cell culture media, leucine and isoleucine were identified as the next critical amino acids due to their limited solubility. This work sought to investigate highly soluble and readily bioavailable derivatives of both amino acids that may be used in batch, fed-batch or perfusion processes. The α-keto acids of Leu and Ile, keto leucine and keto isoleucine sodium salts, were tested in cell culture media and proved to be beneficial to increase the overall solubility of cell culture media formulations. These keto acids were readily bioavailable for various CHO cells and can be used in both media and feeds. The quality of the final recombinant protein was studied in processes using the precursors and the mechanism of amination was investigated in CHO cells. Altogether, both keto acids represent an alternative to their respective amino acids to develop highly concentrated cell culture media formulations to support next generation bioprocesses.